Interactions of Jujube Witches' Broom Phytoplasma Effector with Ziziphus jujuba Host Target: in vitro and in vivo Analyses

Jujube is an important non-wood tree species and fruit. Jujube witches' broom is caused by phytoplasma, which seriously threatens the high-quality development of the jujube industry. Previous studies have shown that the effector Zaofeng6 secreted by phytoplasma interacts with the target protein ZjTCP7 in jujube to induce witches' broom symptoms. This study used subcellular localization, co-immunoprecipitation (Co-IP) and pull-down experiments to verify the direct binding relationship between Zaofeng6 and ZjTCP7 and clarify their molecular interaction mechanism. Online biological software was used to analyze the physicochemical properties and structures of Zaofeng6 and ZjTCP7 proteins, and plant expression vectors and <italic>Escherichia coli</italic> prokaryotic expression vectors of <italic>Zaofeng6</italic> and <italic>ZjTCP7</italic> were constructed. Subcellular localization experiments were performed by transient expression in tobacco, and immunoprecipitation experiments were performed by co-expression in tobacco leaves to verify the interaction of the two proteins in plant cells. Protein expression was induced in <italic>E. coli</italic>, and pull-down experiments were performed to verify the interaction of the two proteins <italic>in vitro</italic>. The result showed that the molecular weights of Zaofeng6 and ZjTCP7 were 9.3426 and 49.6553 kDa, respectively. Zaofeng6 was a hydrophilic stable protein, while ZjTCP7 was a hydrophilic unstable protein. Subcellular localization results showed that Zaofeng6 was localized in the cell membrane and nucleus, while ZjTCP7 was localized in the nucleus. The two proteins were co-localized in the nucleus. Immunoprecipitation experiments showed that GFP-ZjTCP7 signals could be detected in the FLAG antibody immunoprecipitation complex, indicating that the two proteins specifically bind in plant cells. Pull-down experiments further confirmed that GST-ZjTCP7 could specifically adsorb His-Zaofeng6, indicating that the two proteins directly bind <italic>in vitro</italic>. It's concluded that phytoplasma effector factors Zaofeng6 and jujube ZjTCP7 interact in the nucleus, and these two proteins are capable of binding both in planta and <italic>in vitro</italic>. This study lays the foundation for further analysis of the characteristic structures of interacting proteins, as well as for developing targeted control strategies against plant diseases and advancing gene editing research.